A multivalent vaccination strategy for the prevention of Old World arenavirus infection in humans

Abstract

Arenaviruses cause severe human disease ranging from aseptic meningitis following lymphocytic choriomeningitis virus (LCMV) infection to hemorrhagic fever syndromes following infection with Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Machupo virus (MACV), Sabia virus (SABV), or Whitewater Arroyo virus (WWAV). Cellular immunity, chiefly the CD8(+) T-cell response, plays a critical role in providing protective immunity following infection with the Old World arenaviruses LASV and LCMV. In the current study, we evaluated whether HLA class I-restricted epitopes that are cross-reactive among pathogenic arenaviruses could be identified for the purpose of developing an epitope-based vaccination approach that would cross-protect against multiple arenaviruses. We were able to identify a panel of HLA-A*0201-restricted peptides derived from the same region of the glycoprotein precursor (GPC) of LASV (GPC spanning residues 441 to 449 [GPC(441-449)]), LCMV (GPC(447-455)), JUNV (GPC(429-437)), MACV (GPC(444-452)), GTOV (GPC(427-435)), and WWAV (GPC(428-436)) that displayed high-affinity binding to HLA-A*0201 and were recognized by CD8(+) T cells in a cross-reactive manner following LCMV infection or peptide immunization of HLA-A*0201 transgenic mice. Immunization of HLA-A*0201 mice with the Old World peptide LASV GPC(441-449) or LCMV GPC(447-455) induced high-avidity CD8(+) T-cell responses that were able to kill syngeneic target cells pulsed with either LASV GPC(441-449) or LCMV GPC(447-455) in vivo and provided significant protection against viral challenge with LCMV. Through this study, we have demonstrated that HLA class I-restricted, cross-reactive epitopes exist among diverse arenaviruses and that individual epitopes can be utilized as effective vaccine determinants for multiple pathogenic arenaviruses.

FIG. 1.

Cross-reactive in vivo killing of peptide-pulsed target cells in LCMV-infected HLA-A*0201 mice. HLA-A*0201 mice were inoculated by i.p. injection with 2 × 10⁵ PFU of LCMV strain Armstrong 53b or were uninfected (control). Seven days later, CFSE-labeled target cells (CFSE^hi, pulsed with LASV GPC_441-449, LCMV GPC_447-455, JUNV GPC_429-437/MACV GPC_444-452, GTOV GPC_427-435, or WWAV GPC_428-436; CFSE^lo, pulsed with an irrelevant peptide) were delivered to recipient mice by i.v. injection. After 18 h, CFSE-labeled cells were recovered from the spleens of recipient mice and enumerated. Numbers represent the percentage of CFSE^hi peptide-pulsed target cells that were killed in infected animals compared to that in uninfected control animals.

FIG. 2.

Immunization of HLA-A*0201 mice with arenavirus epitopes induces cross-reactive CD8⁺ T-cell responses to heterologous peptides. HLA-A*0201 mice were immunized s.c. with either LASV GPC_441-449 (A), LCMV GPC_447-455 (B), JUNV GPC_429-437/MACV GPC_444-452 (C), GTOV GPC_427-435 (D), WWAV GPC_428-436 (E), or PICV GPC_455-463 (F). Splenic CD8⁺ T cells were isolated 11 to 14 days later and exposed to JA2.1 cells that had been pulsed with serial 20-fold gradient doses (range, 1 × 10⁻⁵ to 6.25 × 10⁻¹¹ M) of either the immunizing peptide or the corresponding heterologous peptides in an ex vivo IFN-γ ELISPOT assay. Peptides were considered immunogenic if they induced IFN-γ spot formation that was significant compared to that on JA2.1 target cells that had been pulsed with an irrelevant HLA-A*0201-restricted peptide. In each graph, the green square/line represents the immunizing peptide, the black triangle/line represents the irrelevant HLA-A*0201-restricted peptide, and the horizontal dashed line indicates the maximal level of IFN-γ spot formation in response to the irrelevant HLA-A*0201 peptide.

FIG. 3.

CD8⁺ T-cell recognition of naturally processed peptides from native LASV GPC or LCMV GPC expressed in HLA-A*0201-restricted human APC. HLA-A*0201 mice were immunized with either LASV GPC_441-449 (A) or LCMV GPC_447-445 (B) as indicated above each graph. Splenic CD8⁺ T cells were isolated 11 to 14 days later and exposed to JA2.1 cells that had been pulsed with peptide (LASV GPC_441-449, LCMV GPC_447-445, or irrelevant peptide) or infected with either wild-type VV (vvWT) or an rVV that expresses the native LASV GPC (vvLASV-GPC) or LCMV GPC (vvLCMV-GPC) in an ex vivo IFN-γ ELISPOT assay. Peptides or rVVs were considered immunogenic if they induced IFN-γ spot formation that was significant compared to that on JA2.1 target cells that had been pulsed with an irrelevant HLA-A*0201-restricted peptide or infected with vvWT. Immunogenic responses are denoted by an asterisk.

FIG. 4.

Heterologous in vivo killing of LASV GPC_441-449- or LCMV GPC_447-455-pulsed target cells in peptide-immunized HLA-A*0201 mice. HLA-A*0201 mice were immunized with LASV GPC_441-449, LCMV GPC_447-455, or adjuvant alone (control). Ten days later, CFSE-labeled target cells (CFSE^hi, pulsed with LCMV GPC_447-455 or LASV GPC_441-449; CFSE^lo, pulsed with an irrelevant peptide) were delivered to recipient mice by i.v. injection. After 18 h, CFSE-labeled cells were recovered from the spleens of recipient mice and enumerated. Numbers represent the percentage of CFSE^hi peptide-pulsed target cells that were killed in arenavirus peptide-immunized animals compared to that in control animals.

FIG. 5.

Peptide immunization of HLA-A*0201 mice with LASV GPC_441-449 provides heterologous protection against a subsequent challenge with LCMV. The logistics of the challenge experiment are outlined in panel A. HLA-A*0201 mice (n = 5 per group) were immunized with adjuvant alone (control), LASV GPC_441-449, or LCMV GPC_447-455 as described in Materials and Methods. On day 14 (d14) postimmunization, mice were inoculated i.p. with 2 × 10⁵ PFU of LCMV strain Armstrong 53b. Spleens were harvested on day 4 postchallenge for determination of viral titer (B) and enumeration of epitope-specific CD8⁺ T cells (C). Mean viral titers from each peptide-immunized group were compared to those of the control group by using the Student t test to determine whether differences were significant. Significant reductions in viral titer are indicated (*, P = 0.005; **, P = 0.004). In panel C, splenic CD8⁺ T cells from each group of animals were exposed to LCMV GPC_447-455, LASV GPC_441-449, or an irrelevant HLA-A*0201-restricted peptide as a control in an ex vivo IFN-γ ELISPOT assay. SE, standard error.